【作者】 郑少萍；

【导师】 谢明星；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2009， 博士

【摘要】 现已证实,肿瘤的生长和发生转移都与其血管的生成具有极高相关性。成纤维细胞生长因子Ⅰ型受体(Fibroblast growth factor receptorl, FGFR1)标志着肿瘤血管已生成,为其标志性分子之一。FGFR1主要通过与其高亲和性配体-bFGF结合来发挥其生物活性作用。可见,阻断bFGF/FGFRl间的信号传导通路就有可能阻断肿瘤血管的形成,进而抑制肿瘤的生长。国外学者和我们前期研究均证实以FGFR1为分子靶标的主动或被动免疫治疗可以有效抑制肿瘤血管生成,达到治疗肿瘤的目的。因此,FGFR1作为抗肿瘤血管生成和靶向治疗的理想靶点,可以进一步应用于后续抗肿瘤血管生成的研究。吉西他滨为嘧啶类抗肿瘤药,对多种实体肿瘤治疗有效。有研究表明应用传统的化疗药物小剂量长期治疗同样具有抗肿瘤血管生成作用,而且没有毒副作用。因此,如果利用主动免疫疗法联合小剂量化疗将有可能取得更好的肿瘤治疗效果。微血管密度(microvessel density, MVD)能较好地反映血管生成活性,但其无法在体实时检测。高频超声结合eFlow显像技术敏感性、时间及空间分辨率高,明显改善了低速和微小血管的显示,使得探测肿瘤微血管成为可能。近年来,超声造影剂及相关技术的应用为超声检查提供了新的诊断方式,极大地改善了超声对血流显示的敏感性。灰阶谐波超声造影技术是近年新兴的一种微循环血流灌注评价方法。造影剂微气泡可增加声波的反射,显著增强二维和多普勒超声的信号,使肿瘤内血流的显示率提高,使造影前未能显示的低速细小血流得以显示。综上,本研究拟在前期研究基础上,利用鸡FGFR1重组蛋白疫苗联合小剂量吉西他滨化疗在小鼠肿瘤模型中进行抗肿瘤血管生成作用观察,了解联合治疗是否能够提高抗肿瘤效应,并初步探讨其作用机理。同时,通过对肿瘤微血管的CDFI、高频超声结合eFlow技术及超声造影的显像结果进行全面定量研究,初步探讨FGFR1重组蛋白疫苗联合吉西他滨抗小鼠肿瘤血管生成效果及其超声成像技术的评价价值。本研究分为以下三部分：本部分在前期研究基础上,以小鼠纤维肉瘤MethA肿瘤模型为对象进行实验,观察抗肿瘤治疗后肿瘤体积、微血管密度、细胞凋亡和增殖以及是否产生抗自身FGFR1抗体等指标,分为联合C+G组、单独cFR组、吉西他滨G单独组、生理盐水NS组,一共4组,了解鸡FGFR1重组蛋白疫苗主动免疫联合小剂量的吉西他滨化疗是否能够提高抗肿瘤效果,并初步探讨其作用机理,为肿瘤治疗开辟新途径。结果显示：①于接种肿瘤后27天,各实验组小鼠肿瘤体积大小(mmm3)分别为876±46.78(C+G组)、1123±82.12(cFR组)、1689±160.11(G组)和4321±182.80(NS组)。联合治疗C+G组与其余各组相比,差异具有统计学(P<0.05)。②C+G组小鼠生存时间较cFR组、G组和NS对照组明显延长,至接种瘤细胞后第46天存活率为90%,C+G组与各对照组小鼠的存活率之间差异具有统计学意义(P<0.05)。③Western blot及免疫荧光检测均发现C+G组、cFR组均有抗FGFR1抗体产生,G组、NS组均未见抗体产生。④ELISPOT检测C+G组、G组、cFR组和NS组小鼠所产生的分泌抗小鼠FGFR1抗体的B淋巴细胞数量计数分别为(113.75±11.08)／106个脾细胞、(5.62±1.79)／106个脾细胞、(112.26±12.47)／106个脾细胞和(5.51±1.92)／106个脾细胞。⑤肿瘤组织内微血管密度计数明显减少,每高倍镜视野各组肿瘤组织内微血管密度分别是9.41±1.78(C+G组)、15.51±3.20(cFR组)、31.88±3.59(G组)和39.25±2.86(NS组),C+G组在分别较之G组、NS组显示差异具有统计学意义(P<0.05)。⑥TUNEL细胞凋亡检测法及PCNA抗体染色方法分别检测结果显示：C+G组、cFR组、G组和NS组各组肿瘤细胞的凋亡指数分别是28.77±3.33%、13.75±1.74%、14.69±2.30%和2.34±0.78%；C+G组凋亡指数明显高于其他各组(P<0.05)。各组增殖指数分别是7.72±1.35%(C+G组)、14.46±3.60%(cFR组)、17.87±3.18%(G组)和28.26±2.33%(NS组),C+G组肿瘤细胞的增殖指数明显低于其他各组(P<0.05)。⑦联合治疗在肿瘤体积、微血管密度、细胞调亡和增殖中均具有协同作用,协同指数分别为1.63、1.05、1.17和1.20。本部分以小鼠纤维肉瘤MethA肿瘤模型为对象进行实验观察,同第一部分分为4组,了解在评价鸡FGFR1重组蛋白联合小剂量吉西他滨抗小鼠肿瘤血管生成疗效中eFlow技术的价值。结果显示：①荷瘤后肿瘤40只小鼠均成活,可见皮下明显的多呈凸外性肿瘤结节。②高频超声结合eFlow技术显示肿瘤内的微血管走行扭曲,管径粗细及血管分布不均匀,可见部分中心向边缘细小血管分支及吻合支。eFlow成像较CDFI更能显示肿瘤内分支细小血管,敏感性更高。③各实验组的血管-肿瘤像素比(%)分别为14.13±3.31(NS),12.11±1.91(G),6.12±1.85(cFR),3.41±1.51 (C+G),呈递减趋势,各组分别与NS组两两相比较,C+G组、cFR组均小于NS组,差异具有统计学意义(P<0.05),G组与NS组间无差异(P>0.05)；C+G组、cFR组、G组组间比较,差异均具有统计学意义(P<0.05)。本部分以小鼠纤维肉瘤MethA肿瘤模型为对象进行实验观察,同第一、二部分分为4组,探讨在评价鸡FGFR1重组蛋白联合小剂量吉西他滨抗小鼠肿瘤血管生成疗效中的超声造影技术的应用价值。结果显示：①造影前小鼠肿瘤内部为不均质回声表现,肿瘤边界显示欠清,CDFI显示肿瘤内部及边缘血供稀疏；注射造影剂后可见小鼠自体血管的血流快速散流向肿瘤周边、在肿瘤边缘绕行后流入肿瘤内部；肿瘤内部实质回声整体有不同程度增强,使得肿瘤边界显示清晰,可视性好。②造影前各组彩色多普勒血管-肿瘤像素比分别为C+G组(1.71±1.34)、cFR组(3.81±1.99)、G组(5.62±2.22)、NS组(9.12±2.77)；造影后各组彩色多普勒血管-肿瘤像素比分别为C+G组(5.91±1.37)、cFR组(9.52±1.35)、G组(15.51±2.42)、NS组(20.72±3.23),造影前后C+G组、cFR组、与NS组间血管-肿瘤像素比差异具有统计学意义(P<0.05)；G组与NS组间差异无统计学意义(P>0.05)；造影前后同一组内差异的比较具有统计学意义(P<0.05)。1.鸡FGFR1重组蛋白疫苗联合低剂量吉西他滨抗肿瘤血管生成的作用比单独治疗更佳；联合治疗抗肿瘤作用主要是通过主动免疫诱导产生抗自身FGFR1的抗体抑制肿瘤血管生成来实现的；联合治疗在抑制肿瘤生长及肿瘤血管生成、诱导肿瘤细胞凋亡和抑制细胞增殖作用中具有协同效应。2.高频超声结合eFlow显像技术能够显示肿瘤内的微血管走行、分布情况,较CDFI更能显示肿瘤内分支细小血管,敏感性更高,为评价小鼠肿瘤模型中抗肿瘤血管生成的疗效提供了一种实时的、无创性的新方法。3.超声造影可增强肿瘤内微血管的走行、分布的显示及肿瘤与其相邻组织之间的对比性,从而提高肿瘤及肿瘤微血管的检出率,能够为在体实时评价小鼠肿瘤模型中抗肿瘤血管生成效果提供一种直观、敏感的方法。更多还原

【Abstract】 It is well known that the tumor growth and metastasis are closely related to tumor angiogenesis. Fibroblast growth factor receptor 1 (FGFR1) is one of the most important angiogenesis factor, and plays a role in its biological activity primarily through its high-affinity and ligand-bFGF. Therefore, blocking the signal transduction pathway between the bFGF/FGFR1 can inhibit the formation of tumor blood vessels, and sequently suppress tumor growth. Foreign scholars and our preliminary studies confirmed that the active or passive immunotherapy targeting FGFR1 can effectively inhibit tumor angiogenesis to achieve the purpose of tumor treatment. Accordingly, FGFR1 are regarded as the ideal target for anti-tumor angiogenesis and targeted therapy and can be used for further anti-cancer research.Gemcitabine is a new deoxycytidine analogue and has shown cytotoxicity against solid tumors. Studies have shown that traditional low-dose chemotherapy for a long time has the same effect on the treatment of anti-angiogenesis but no toxic side-effects. Thus, it is possible to achieve better treatment of tumor by combining the active immunotherapy with low-dose chemotherapy.Microvessel density (MVD) can better reflect the angiogenesis activity, however, its can not be detected in vivo. Eflow combination with high-frequency ultrasound imaging is more sensitive and has more time and high spatial resolution, which significant manifests the low-speed flow and small blood vessels, making it possible to detect the tumor microvessel. In recent years, the application of ultrasound contrast agents and its related technology provides a new way for ultrasound diagnosis, and greatly improved the diagnostic sensitivity of the blood flow. Harmonic gray-scale contrast-enhanced ultrasound technology is a new method to evaluate the blood flow microcirculation in recent years. Gas microbubble of the contrast agent can increase the acoustic reflex, and significantly enhance the two-dimensional image and Doppler ultrasound signal, improving the display rate of tumor blood flow and manifesting the low blood flow that can not show before ultrasound contrast.Taken together, we investigated the anti-tumor angiogenesis effect of combining the chicken FGFR1 recombinant protein vaccine with low doses of gemcitabine chemotherapy in mouse tumor model, and explored the related mechanism. Meanwhile, we studied the function of the CDFI, high-frequency ultrasound combined with eflow and ultrasound contrast imaging technology in quantitatively detecting tumor microvessel, and investigated the role of ultrasound imaging technology in evaluating the treatment of anti-tumor angiogenesis by chicken FGFR1 recombinant protein vaccine combined with low-dose-gemcitabine.This study is divided into three parts as follows:This part is based on the preliminary studies. we aim the MethA fibrosarcoma tumor model in mice as the object for experimental observation about the tumor volume, microvessel density, apoptosis and proliferation and whether they could produce their own anti-FGFR1 antibody or not after anti-tumor treatment. All the mice were divided into 4 Groups:combined therapy group (C+Ggroup), recombinant protein vaccine of chicken FGFR1 alone treatment group (cFR group), gemcitabine alone treatment group (G group), non-treatment control group (NS group), to know how the chicken FGFR1 recombinant protein active immunization vaccine combined with low-dose gemcitabine chemotherapy can improve the anti-tumor effects and to explore its mechanism, so that we can open up a new way of tumor therapy.Results:①In the 27th, after tumor inoculation, the tumor size (mm3) of the mice were 876±46.78 (C+G group),1123±82.12 (cFR group),1689±160.11 (G group) and 4321±182.80 (NS group). C+G combination therapy group made statistical difference compared with the rest of the groups (P<0.05).②C+G group was significantly prolonged the survival time of mice. After inoculation tumor cells for 46 days the survival rate of C+G group was 90%, C+G group and the control group, the difference between the survival rate of mice with statistical significance (P＜0.05).③Western blot and immunofluorescence assay have found that it have generated anti-FGFR1 antibody in C+G group and cFR groups. Without any anti-FGFR1 antibody has been found in G group and NS group.④ELISPOT detection the number of C+G group, cFR group, G group and the NS group of the B-lymphocyte which can generate anti-mouse-FGFR1 antibody, are (113.75±11.08)/106, (112.26±12.47)/106, (5.62±1.79)/106, (5.51±1.92)/106.⑤The count of tumor microvessel density in high-power microscope field of vision of each group were 9.41±1.77 (C+G group),15.5±3.20 (cFR group),31.9±3.60 (G group) and 39.3±2.87 (NS group). Among C+G group, G group and NS group, the differences were significant (P<0.05).⑥TUNEL and PCNA antibody staining was detected the tumor cells apoptosis and proliferation. The results showed that:tumor cells apoptotic index of each group were 28.77±3.33% (C+G group),13.75±1.74% (cFR group),14.69±2.30%(G group) and 2.34±0.78% (NS group). C+G group apoptotic index was significantly higher than other groups (P＜0.05). C+G group of tumor cell proliferation index was significantly lower than other groups (P＜0.05), proliferation index in each group were 7.72±1.35% (C+G group), 14.46±3.60% (cFR group),17.87±3.18% (G group) and 28.26±2.33% (NS group).⑦Combination therapy in tumor volume, microvessel density, apoptosis and proliferation are synergies, and the synergy index is 1.63,1.05, 1.17 and 1.20.In this part, we aim the MethA fibrosarcoma tumor model in mice as the object for experimental observation. All the mice are divided into 4 groups: treatment group (C+G group), chicken FGFR1 recombinant protein vaccine alone treatment group (cFR group), gemcitabine alone treatment group (G group), non-treatment control group (NS group), to exploring the evaluation value of color Doppler imaging and high-frequency ultrasound combined with eFlow imaging for detecting the efficacy of anti-tumor angiogenesis by chicken FGFR1 recombinant protein combined gemcitabine.Results:①40 mice were all successed to be tumor-beared (40/40). The tumor formations are obvious subcutaneous nodules.②Most of the nodules are prominent growth out, the early type was spherical, the latter was the disc-shaped or lobulated.③It is visible to see the large distortion trophoblast vascular of the tumor on the surface of the lower skin. High-frequency ultrasound combined with eFlow Technology showed that tumor microvessel traveling distorted, and the vascular diameter of uneven thickness, we can see some of the centers to the edge of a small vascular branches and ansa. eFlow imaging can show more small branches of tumor blood vessels, and has a higher sensitivity than CDFI.④The vessels-tumor-pixel ratio (%) of C+G group, cFR group, G group, NS Group were 3.41±1.51,6.12±1.85, 12.11±1.91,14.13±3.31, and the trend was higher. Compared with the NS group, either C+G group or cFR group are less than it, with the statistically significant differences (P＜0.05). There was no significant difference between G group and NS group (P>0.05); C+G group, cFR group, G group of inter-group differences were statistically significant (P＜0.05). In this part, we aim the MethA fibrosarcoma tumor model in mice as the object for experimental observation. All the mice were divided into 4 groups: treatment group (C+G group), chicken FGFR1 recombinant protein vaccine alone treatment group (cFR group), gemcitabine alone treatment group (G group), non-treatment control group (NS group), to explore the evaluation value of the effect of chicken FGFR1 recombinant protein combined low-dose gemcitabine in anti-tumor angiogenesis by ultrasound contrast imaging.Results:①Before ultrasound contrast, the tumor 2D echo showed less clearance edge, and CDFI showed that the tumor blood flow within and around being the scarce supply. After contrast-enhanced imaging, the host of blood vessels in tumors can be seen rapid flow bypass and into the tumor after injecting the contrast agents about 3-8s, and the tumor echo showed varying degrees, in real terms and in the overall enhanced which can make the tumor border more clear to see.②Before contrast, color Doppler blood vessels-tumor pixels ratio (%) in C+G组each group were C+G group (1.71±1.34), cFR group (3.81±1.99), G group (5.62±2.22), NS group (9.12±2.77). After contrast imaging, the vascular-tumor pixels in each group were C+G group (5.91±1.37), cFR group (9.52±1.35), G group (15.51±2.42) and NS group (20.72±3.23). Before and after contrast, the blood vessels-tumor pixels of C+G group, cFR group, NS group have statistical significance differences (P<0.05); G group and NS group there was no significant difference (P＞0.05); within the same group, also there is a statistically significant difference (P<0.05).ConclusionBased on the present study, our conclusions are as follows:①The angiogenesis effect of Chicken FGFR1 recombinant protein vaccine combined with low-dose gemcitabine antitumor is better than medication alone; the role of combined anti-tumor active immunization mainly through self-induced anti-FGFR1 antibody to inhibite tumor angiogenesis; Combined therapy have a synergistic effect on inhibiting tumor growth and tumor angiogenesis, tumor-induced apoptosis and inhibited cell proliferation. ②High-frequency ultrasound combined with eFlow imaging technology can show tumor microvessel course and the distribution of small branches of tumor blood vessels, which has a higher sensitivity than CDFI. It can provide a real-time and a new non-invasive method for the evaluation of anti-tumor effect of angiogenesis.③Ultrasound contrast may enhance the tumor and the tumor microvessel display contrast with the surrounding tissue, and enhance the tumor and the tumor microvessel detection rate. It can provide an intuitive, sensitive and real-time methods for evaluation the anti-tumor effect of angiogenesis.更多还原